The COVID-19 pandemic, caused by SARS-CoV-2, necessitates the development of effective therapeutics. Current treatment options are limited, highlighting the urgent need for readily available and easily administered preventative and treatment strategies. This research investigated the antiviral potential of sulforaphane (SFN), a naturally occurring compound found in cruciferous vegetables, known for its antioxidant and anti-inflammatory properties. SFN's ability to activate the NRF2 transcription factor, enhance macrophage activity, and reduce lung inflammation in preclinical models suggested its potential antiviral activity against both seasonal coronaviruses (like HCoV-OC43) and SARS-CoV-2. The study hypothesized that SFN could provide antiviral activity against both HCoV-OC43 and SARS-CoV-2, building upon prior research in animal models using attenuated influenza viruses and demonstrating favorable pharmacokinetics and low toxicity in preclinical trials. The researchers screened readily available drugs for efficacy against human coronaviruses, starting with HCoV-OC43 and then testing promising candidates against SARS-CoV-2.

The introduction thoroughly reviews the limitations of current COVID-19 treatments, emphasizing the need for easily accessible and effective therapeutics. It cites existing literature on the antiviral properties of sulforaphane (SFN) and its known mechanisms of action, such as NRF2 activation and anti-inflammatory effects. The review also highlights previous research on SFN's efficacy against other viruses, setting the stage for its investigation against SARS-CoV-2 and HCoV-OC43. This lays the groundwork for the study's hypothesis and experimental design.

The study employed both in vitro and in vivo methods. In vitro experiments used Vero C1008 and MRC-5 cells (for HCoV-OC43) and Caco-2 cells (for SARS-CoV-2). Cells were exposed to SFN before or after virus inoculation, and viral replication was assessed via cytopathic effect (CPE) assays and qPCR. Synergistic effects with remdesivir were evaluated using combination assays. The in vivo study utilized K18-hACE2 transgenic mice, which express human ACE2, allowing SARS-CoV-2 infection. Mice received daily oral SFN before intranasal SARS-CoV-2 infection. Viral load, lung injury (measured by BAL fluid protein), and pulmonary pathology were assessed. Immune cell populations in lung and spleen tissues were analyzed using high-dimensional flow cytometry. Statistical analyses included one-way and two-way ANOVA, Mann-Whitney U tests, and calculation of combination indices (CI). Specific techniques included H&E staining, immunostaining for SARS-CoV-2 spike protein, and qPCR for viral RNA quantification. NRF2 knockdown experiments were performed in Caco-2 cells to investigate the role of NRF2 in SFN's antiviral activity. Detailed descriptions of cell culture, virus strains, drug preparation, assays, and statistical analyses are provided in the methods section.

In vitro, SFN inhibited replication of HCoV-OC43 and multiple SARS-CoV-2 strains (including Delta and Omicron) in a dose-dependent manner. The median inhibitory concentrations (IC50) varied across cell lines and virus strains but were within the micromolar range. SFN demonstrated a synergistic interaction with remdesivir in inhibiting viral replication. In vivo, prophylactic oral SFN treatment in K18-hACE2 mice significantly reduced lung viral load (1.5 log reduction), total protein in bronchoalveolar lavage fluid (indicating reduced lung injury), and pulmonary pathology. SFN treatment also modulated the immune response, reducing myeloid cell recruitment to the lungs and decreasing T cell activation and cytokine production. Interestingly, the antiviral activity of SFN appeared to be independent of NRF2 activation in vitro.

The findings demonstrate SFN's broad-spectrum antiviral activity against both seasonal and pandemic coronaviruses, both in vitro and in vivo. The synergistic interaction with remdesivir suggests potential for combination therapies. The observed reduction in lung injury, pulmonary pathology, and immune cell activation highlights SFN's multi-faceted mechanism of action. The in vitro results suggest that the mechanism of action may be independent of NRF2 modulation, while the in vivo findings highlight the role of NRF2 in the anti-inflammatory effects. These results are significant because they identify a readily available and relatively safe compound with potential for preventing or treating coronavirus infections. The study's findings support further investigation into SFN's potential as a therapeutic agent for COVID-19 and other respiratory viral infections. The clinical relevance of SFN's in vitro efficacy is supported by the observation that the peak plasma concentrations achievable after consuming SFN-rich foods are within the micromolar range of the IC50 values observed in the study.

This study demonstrates that sulforaphane (SFN) exhibits potent antiviral activity against SARS-CoV-2 and HCoV-OC43 both in vitro and in vivo, with a potential synergistic effect when combined with remdesivir. The observed reduction in lung inflammation and immune dysregulation further supports SFN's therapeutic potential. Future research should focus on clinical trials to evaluate SFN's efficacy and safety in humans, as well as further mechanistic studies to fully elucidate its antiviral and immunomodulatory properties. Investigating optimal dosing strategies and formulations for maximizing bioavailability and therapeutic benefits in humans is also crucial.

The study used a transgenic mouse model (K18-hACE2) which may not fully recapitulate human infection. Only male mice were used, limiting generalizability. The bioavailability of SFN can vary depending on several factors, including individual gut microbiota, which could influence the results. Further research is needed to fully characterize the mechanism of action and to assess the clinical relevance of these findings in diverse human populations.